The BRCA1 Tumor Suppressor Binds to Inositol 1,4,5-Trisphosphate Receptors to Stimulate Apoptotic Calcium Release*

Background: The non-nuclear BRCA1 tumor suppressor can stimulate cell death, but the mechanisms are unknown. Results: BRCA1 binds to the inositol 1,4,5-trisphophate receptor (IP3R) calcium channel at the endoplasmic reticulum to stimulate apoptotic calcium release. Conclusion: BRCA1 tumor suppressor activity includes direct stimulation of apoptotic cell death via increased IP3R activity. Significance: We identify a novel role for the tumor suppressor BRCA1. The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.

The IP 3 R 2 is a ligand-gated calcium channel localized primarily to ER membranes. The IP 3 R is a tetrameric protein, and each subunit consists of an N-terminal ligand binding domain, a C-terminal transmembrane pore domain, and an intervening modulatory domain. Calcium release through the IP 3 R regulates multiple cellular processes, including, but not limited to, gene expression, metabolism, and apoptosis (1,2). Numerous IP 3 R protein-protein interactions allow for signal integration of diverse signaling pathways (1). Several proto-oncogenes and tumor suppressors interact with and regulate the IP 3 R, including Bcl-2, PTEN, and PML (3)(4)(5). These proteins regulate IP 3 R activity and apoptotic calcium release through multiple mechanisms. In general, proto-oncogenes cause reduced IP 3 R activity, whereas tumor suppressors cause increased IP 3 R activity to regulate cell death (4).
Breast and ovarian cancer susceptibility gene 1 (BRCA1) is a tumor suppressor well known for its function during homologous recombination and repair of DNA double strand breaks (6 -8). The presence of nuclear export and import signals suggests a regulated transport of BRCA1 into and out of the nucleus (9,10). Several studies have suggested a pro-apoptotic role for BRCA1 linked to its nuclear export and cytosolic localization, but the mechanisms by which BRCA1 stimulates cell death outside of the nucleus are unclear (11)(12)(13).
Here we show that BRCA1 stimulates apoptosis through a physical and functional interaction with the IP 3 R. BRCA1 is recruited to the IP 3 R during apoptosis and stimulates apoptotic calcium release and cell death. Furthermore, we identified a pool of BRCA1 that is constitutively localized to the ER via binding of the BRCA1 C terminus (BRCT) domain to phospholipids, which may facilitate the rapid recruitment of BRCA1 to the IP 3 R during cell death. Therefore, BRCA1 mediates its proapoptotic effects by binding to and modulating apoptotic calcium release through the IP 3 R. lain with 326 l of buffer B (2.3 M sucrose, 10 mM HEPES (pH 7.5), 2 mM MgCl 2 ) and centrifuged for 1 h at 40,000 rpm. The supernatant was removed by aspiration. P1 pellets were resuspended in buffer A. Resuspended pellets were sonicated in a bath sonicator in ice water for 30 min before being passed through a 23-gauge needle 10 times to shear genomic DNA.
Cell Death Assays-Propidium iodide and caspase-3 measurements were performed as described previously (16).
Cytosolic Calcium Imaging-HeLa cells were transfected with full-length YFP-BRCA (9). After 48 h, cells were loaded with 2.5 M Fura-2 in imaging buffer composed of 1% BSA, 107 mM NaCl, 20 mM HEPES, 2.5 mM MgCl 2 , 7.25 mM KCl, 11.5 mM glucose, 1 mM CaCl 2 for 30 min at room temperature. The solution was replaced with imaging solution without Fura-2 for an additional 30 min prior to imaging. Coverslips were then imaged on a Nikon TiS inverted microscope with a ϫ40 oil objective as described previously (17). Responses to 100 nM, 1 M, and 10 M histamine were recorded in YFP-BRCA1 cells and adjacent YFP-negative control cells from four separate coverslips. A total of 25 individual YFP-BRCA1-positive and 24 YFP-negative cells were quantified and pooled from the four coverslips. Peak release was determined in the MetaFluor acquisition software. Oscillation frequency was determined manually, where an oscillation was defined as a rise and fall of the Fura-2 ratio of at least 0.01 units. This same threshold was used to determine the percentage of responding cells at 100 nM histamine. All cells responded at 1 and 10 M histamine and, therefore, were not quantified. For the siRNA knockdown experiments, a similar approach was used. HeLa cells were transfected with two different siRNAs targeting human BRCA1 (Ambion/Life Technologies siRNAs s458 and s459). The total amount was 12.5 pmol/well of a 6-well dish. Transfected cells were identified by cotransfection with YFP. Cells were imaged after 48 h. We found that both siRNAs efficiently knocked down BRCA1 expression (Fig. 3A). However, siRNA s458 also up-regulated IP 3 R-1 expression (Fig. 3A). Therefore, only siRNA s459 was used for the calcium imaging experiments. The siRNA data were quantified exactly as above, with the exception that the control cells were transfected with control siRNA (control siRNA, medium GC content, Life Technologies, catalog no. 12935-300) on separate coverslips and identified by YFP expression exactly as described for the BRCA1-targeted siRNA.
Mitochondrial Calcium Imaging-Mitochondrial calcium was quantified by Rhod-2 imaging exactly as described previously (17). The response to 10 M histamine was quantified and pooled from five coverslips comprising 18 YFP-BRCA1 and 18 YFP-negative cells.
ER Calcium Imaging-HeLa cells were loaded with 5 M Mag-Fura-2/AM in imaging solution (1% BSA, 107 mM NaCl, 20 mM HEPES, 2.5 mM MgCl 2 , 7.25 mM KCl, 11.5 mM glucose, and 1 mM CaCl 2 ) for 20 min at room temperature. To obtain cytosolic access and image ER calcium, cells were permeabilized with 120 g/ml saponin in intracellular solution (125 mM KCl, 19 mM NaCl, 10 mM HEPES, 1 mM EGTA, and 0.4 mM CaCl 2 ) until permeabilization was obvious via imaging (ϳ1 min). Images were taken every 3 s during continuous recording of the response to various additions. Measurement of steadystate changes in cytosolic calcium after 24 h of paclitaxel treat-BRCA1 Binds To IP 3 R MARCH 13, 2015 • VOLUME 290 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 7305 ment ( Fig. 4D) was performed using Fura-2 as described previously (15). IP 3 R Single Channel Recording-Recombinant rat IP 3 R-1 was stably expressed in triple IP 3 R knockout DT40 cells. Single channel recording by nuclear patch clamp technique was performed exactly as described previously (18). The recording solution contained 140 mM KCl, 10 mM HEPES, 5 mM NaATP, 100 M BAPTA, 200 nM free calcium, and 1 M IP 3 . Channels were recorded at Ϫ100 mV, sampled at 20 kHz, and filtered at 5 kHz. A minimum of 15 s of recording from patches containing a single channel was used for analysis. The total number of channels analyzed for each condition are indicated over the bar graph in Fig. 4B. Where indicated, either 30 nM BRCA1 GST-RING or 30 nM GST alone was included in the patch pipette.
FRET Imaging-HeLa cells were transfected with YFP-BRCA1 (9) and CFP-IP 3 R (15). After 48 h, cells were imaged on a Nikon TiS inverted microscope with a ϫ100 SuperFluor objective, and images were acquired every 30 s with a Photometrics Evolve EMCCD camera. Excitation and emission filters for acquiring donor and acceptor channels were from Chroma Technology (set 89002). Cells were maintained at 37°C during imaging. Data were quantified by taking a region of interest from the cytosol and nucleus and taking the ratio of the acceptor fluorescence (excitation, 430 nm; emission, 535 nm) over the donor fluorescence (excitation, 430 nm; emission, 470 nm) for each region of interest. The data in Fig. 5B were pooled from 20 cells (CFP-IP 3 R/YFP-BRCA1) and 22 cells (CFP-IP 3 R/YFP) from at least three separate experiments.
Fatty Acid Binding Site Prediction-To screen for a putative fatty acid binding region in BRCA1, a position-specific scoring matrix (PSSM)-based method, described previously in Ko et al. (19), was used. An initial PSSM library was defined from the experimentally determined fatty acid binding protein regions in 42 well characterized lipid binding crystal structures collected from the Protein Data Bank (20 -23). This initial PSSM library was leveraged to search for more fatty acid binding protein regions using psi-blast and, therefore, expanded to 1185 fatty acid binding protein-specific PSSM libraries. Human BRCA1 was aligned with this expanded fatty acid binding protein-specific PSSM library. All positive alignments were recorded. From these positive PSSMs, a residue score was calculated that represents the occurrence of identical and similar residues from each query-PSSM alignment above the threshold. The Smith-Waterman algorithm was used to re-evaluate PSSM comparisons with the query sequence that resulted in a positive alignment, as in 24, 25. Then raw scores for each residue were calculated by scoring a value of 2 for identities and a value of 1 for positive substitutions from each alignment. These values were summed for all alignments at each position to obtain a total raw residue score.

RESULTS
BRCA1 Binds to the IP 3 R-In a yeast-two-hybrid experiment using the C-terminal tail domain of IP 3 R (amino acids 2589 -2749) as bait, the RING domain of BRCA1 was isolated as an interacting clone (amino acids 1-112). To confirm this direct interaction, we used purified recombinant protein in an in vitro binding experiment. The purified RING domain was covalently conjugated to cyanogen bromide-activated agarose beads, and the ability of the purified GST-IP 3 R tail domain and modulatory domain (amino acids 923-1582) to bind to the conjugated beads was tested. The RING domain of BRCA1 was able to specifically pull down the IP 3 R tail domain, suggesting a direct protein-protein interaction between BRCA1 and the IP 3 R tail (Fig. 1A). To test for a physical interaction between full-length BRCA1 and IP 3 R in cells, we performed a coimmunoprecipitation experiment. Using an IP 3 R antibody, we were able to coimmunoprecipitate endogenous IP 3   The asterisk indicates a possible spliceoform of BRCA1 that is detected by the antibody. In our hands, commercial antibodies to BRCA1 were not suitable for reciprocal coimmunoprecipitation. C, subcellular fractionation by differential centrifugation of HeLa cells. BRCA1 expression was detected at the predicted molecular weight (BRCA1) and a slower migrating band that is likely hyperphosphorylated (pBRCA1). The asterisk indicates a possible spliceoform of BRCA1 that is detected by the antibody. Fraction purity was evaluated with the organelle controls lactate dehydrogenase (LDH, cytosol), IP 3 R-1 (endoplasmic reticulum), cytochrome c oxidase (CytCOx, mitochondria), and histone H1 (nucleus). The experiments in B and C utilized HeLa cell lysates.
in HeLa cells (Fig. 1B). We could not perform a reciprocal coimmunoprecipitation of the IP 3 R with BRCA1 because we were unable to identify an antibody suitable for immunoprecipitation of BRCA1. This may be related to epitope masking in Triton X-100 lysates of BRCA1. Regardless, yeast two-hybrid screening, coimmunoprecipitation, and direct binding of purified components strongly suggest a direct physical interaction between BRCA1 and the IP 3 R mediated by the BRCA1 RING domain and the IP 3 R tail domain. The existence of BRCA1 nuclear import and export signals indicates the regulated transport of the protein into and out of the nucleus. However, the subcellular localization of BRCA1 has been a subject of debate (26). To determine the subcellular localization of BRCA1, we performed subcellular fractionation using differential centrifugation of HeLa cell homogenates. We used HeLa cells because IP 3 R function, subcellular localization, and protein interactions during apoptosis have been studied extensively and are well characterized in this cell line (15,27). Furthermore, HeLa cells express endogenous wild-type BRCA1 (28). After differential centrifugation, the purity of the individual fractions was confirmed by stripping and reprobing the same blot with IP 3 R-1 (endoplasmic reticulum), histone H1 (nucleus), cytochrome c oxidase (mitochondria), and lactate dehydrogenase (cytosol). BRCA1 was expressed in all cell fractions except the 100,000 ϫ g supernatant (representing cytosol), and, surprisingly, the majority of BRCA1 was localized to the 100,000 ϫ g microsomal ER-enriched fraction (Fig. 1C). IP 3 Rs in this fraction also specifically bind to cytochrome c to regulate apoptotic calcium release (15). Interestingly, the phosphorylated form of BRCA1 was also found in the 10,000 ϫ g pellet, which is enriched in IP 3 R and mitochondria, consistent with a previous report (28). Therefore, non-nuclear BRCA1 is abundant and localized to the same subcellular compartments as the IP 3 R and could potentially modulate its activity. BRCA1 Modulates IP 3 R Function-We next chose to examine the effect of BRCA1 on IP 3 R function by expressing fulllength BRCA1 fused to the C terminus of YFP in HeLa cells and examining the response to escalating doses of histamine. HeLa cells were used in these experiments because of their ease of transfection and well characterized calcium release characteristics after histamine challenge (29 -31). The YFP-BRCA1 fusion protein has been characterized previously and is functionally comparable with the wild-type protein (12,32). As shown in Fig. 2A, YFP-BRCA1 is localized to both nuclear and non-nuclear compartments. We measured calcium release in response to 100 nM, 1 M, and 10 M histamine in cells expressing YFP-BRCA1 and adjacent cells not expressing the fusion protein. As shown in Fig. 2, B-E, BRCA1 expression significantly sensitized HeLa cells to a 100 nM histamine challenge, increasing both peak release (Fig. 2, C and D) and the number of responding cells (Fig. 2E). Expression of BRCA1 did not appear to affect peak release of calcium in response to 1 M histamine or a saturating dose of 10 M histamine (Fig. 2, F and G). However, at both of these doses there is a significant increase in the oscillation frequency, which would be expected to have profound implications for downstream signaling events (Fig. 2H) (33,34). Therefore, expression of BRCA1 has significant effects on calcium signaling through IP 3 R-coupled pathways. It has been shown that BRCA1, and the BRCA1 binding protein, and homologue BARD1 are targeted to mitochondria (28,35), and our subcellular fractionation results indicate that a significant amount of BRCA1 is present in a fraction that also contains mitochondria (Fig. 1C). We hypothesized that BRCA1 may facilitate calcium transfer into mitochondria. To test this hypothesis, we stimulated HeLa cells with 10 M histamine and measured calcium uptake into mitochondria using Rhod-2. As shown in Fig. 2I, BRCA1 expression has no effect on calcium uptake into mitochondria.
The above results indicate that BRCA1 has significant effects on IP 3 R-coupled calcium release induced by histamine but does not determine whether BRCA1 has a direct effect on IP 3 R function. To directly measure IP 3 R activity, we used saponin-permeabilized cells loaded with MagFura-2 to measure ER calcium release induced by IP 3 addition to the bath. MagFura-2 imaging of ER stores is a well established method for measuring ER calcium store depletion in response to IP 3 R activation (36,37). Cell membrane permeabilization also allows for access to the cytosol and the ability to directly activate IP 3 R with addition of IP 3 to the bath (38). We found that cells expressing YFP-BRCA1 released significantly more calcium after addition of both subsaturating (200 nM) and saturating (10 M) doses of IP 3 (Fig. 2,  J and K). This indicates that expression of BRCA1 directly increases IP 3 R activity, which is consistent with the functional effects on histamine-induced calcium release.
The experiments in Fig. 2 examined the effects of overexpressed BRCA1 on IP 3 R function. We next wished to determine whether endogenous BRCA1, which we found to bind endogenous IP 3 R-1 (Fig. 1B), also modulates calcium release. We obtained two commercial siRNA duplexes targeting human BRCA1 and determined their efficiency in knocking down endogenous BRCA1 expression in HeLa cells. We found that both siRNA duplexes almost completely abrogated BRCA1 expression in HeLa cells (Fig. 3A). We found that one of the siRNA complexes (s458) also up-regulated IP 3 R-1 expression, which may reflect a compensatory mechanism. Regardless, further experiments were performed with the siRNA duplex that did not affect IP 3 R expression (s459). Knockdown of BRCA1 expression had the opposite effect of overexpression, including decreased peak release, decreased oscillation frequency, and a dramatic reduction in the number of percent responders to 100 nM histamine compared with cells expressing control siRNA (Fig. 3, B-E). In addition, BRCA1 knockdown also decreased peak release to 1 and 10 M histamine (Fig. 3F). Therefore, endogenous BRCA1 has significant effects on the activity of endogenous IP 3 R channels in HeLa cells.
BRCA1 Increases IP 3 R Open Probability-To determine the direct effects of BRCA1 on IP 3 R activity, we recorded singlechannel currents on isolated nuclei that express recombinant rat IP 3 R-1 (Ref. 18 and "Experimental Procedures"). As shown in Fig. 4, A and B, when the BRCA1 GST-RING domain was included in the patch pipette, the open probability of the IP 3 R-1 channel in the presence of a subsaturating concentration of IP 3 (1 M) was increased dramatically (control, 0.21 Ϯ 0.02; GST-RING, 0.57 Ϯ 0.06; GST only, 0.21 Ϯ 0.02). This was due to a destabilization of the closed state of the channel (Fig. 4C).
Therefore, the BRCA1 RING domain directly and potently increases IP 3 R activity by modulating channel gating.
BRCA1 Binds to IP 3 Rs during Cell Death to Stimulate Calciumdependent Apoptosis-We hypothesize that BRCA1 binding to IP 3 R increases during cell death and that this is an essential component of its tumor suppressor activity. To measure changes in the BRCA1/IP 3 R interaction in living cells during apoptosis, we used the FRET pair CFP-IP 3 R and YFP-BRCA1. We used the clinically relevant chemotherapeutic paclitaxel to induce apoptosis and measured dynamic changes in the FRET ratio (and, therefore, binding) in HeLa cells transfected with CFP-IP 3 R-1 and YFP-BRCA1 or YFP alone (Fig. 5A). We delib-erately avoided DNA-damaging chemotherapeutics such as cisplatin so that the DNA repair activity of BRCA1 would not confound the results. We found a significant increase in cytosolic FRET after treatment with paclitaxel (1 M) in cells expressing both CFP-IP 3 R-1 and YFP-BRCA1 (Fig. 5B). The kinetics of association are consistent with the time course of activation of cell death proteins such as JNK1 in response to paclitaxel treatment (39). We saw no increase in FRET after treatment with paclitaxel in the nucleus of cells transfected with CFP-IP 3 R-1 and YFP-BRCA1 (Fig. 5B) or in cells transfected with CFP-IP 3 R-1 and YFP only (Fig. 5C). These results indicate that BRCA1 is recruited to IP 3 R channels on the ER during apoptosis and support the hypothesis that, under resting (i.e. non-apoptotic) conditions, only a subpopulation of IP 3 Rs are bound to BRCA1. We next examined the effect of BRCA1 expression on paclitaxel-induced apoptosis. In these experiments, we used the patient-derived, BRCA1-mutated cell line UWB1.289 (UWB) isolated from an ovarian carcinoma, and this same cell line stably rescued wild-type BRCA1 (UWB-BRCA1). The BRCA1 mutations in the parental UWB line eliminate expression of the BRCA1 protein. Treatment of UWB cells with paclitaxel for 24 h did not cause an elevation of cytosolic calcium, whereas UWB cells with rescued BRCA1 expression had significantly elevated calcium consistent with apoptotic calcium release (Fig.  5D). This indicates that BRCA1 expression is required for paclitaxel-induced apoptotic calcium release via the IP 3 R. Measurement of apoptosis by both caspase-3 enzymatic activity and  propidium iodide staining indicated that BRCA1 expression was required for efficient paclitaxel-induced cell death of ovarian carcinoma cells (Fig. 5, E and F). This suggests that BRCA1 expression restores paclitaxel sensitivity to the BRCA1-null cells. This is consistent with similar findings by other groups (40).

BRCA1 Is Recruited to the ER via IP 3 Rs and a Novel Lipid
Binding Domain-We next examined whether IP 3 R expression is required for BRCA1 localization in non-nuclear compartments. We used DT40 cells as well as DT40 IP 3 R triple knockout (DT40 TKO) cells. This is the only cell line currently available that is deficient in all three IP 3 R isoforms (41). We transiently transfected DT40 and DT40 TKO cells with YFP-BRCA1 and nuclear localized DsRed (Fig. 6A, left panel) and scored cells with nuclear only or nuclear and cytosolic expression in a blinded manner. The number of cells with BRCA1 non-nuclear localization was significantly higher in DT40 cells expressing IP 3 R compared with DT40 TKO cells (Fig. 6A, right   panel). This indicates that IP 3 R expression is a partial determinant of BRCA1 localization outside of the nucleus.
To determine whether BRCA1 is recruited to ER membranes during apoptosis, we treated DT40 and DT40 TKO cells with paclitaxel and purified ER-enriched 100,000 ϫ g fractions as in Fig. 1C. BRCA1 ER localization increased with paclitaxel treatment, and this increase was dependent on IP 3 R expression (Fig.  6B). Interestingly, loss of IP 3 R expression did not completely prevent constitutive ER localization of BRCA1. This suggests that a subpopulation of BRCA1 is associated with the ER independent of both IP 3 R expression and apoptosis. We hypothesized that recruitment of BRCA1 to the ER may be mediated by an intrinsic lipid binding activity in the BRCA1 protein. We identified a potential lipid binding domain within the C-terminal tandem BRCT domain of BRCA1 using Adaptive-BLAST (24,25). Specifically, we found that amino acid residues 1664 -1696 within the first BRCT repeat have a strong potential for lipid binding on the basis of homology to fatty  acid binding proteins ( Fig. 6C and "Experimental Procedures"). Examination of high-scoring residues within this sequence identified a basic region with several threonine residues common to lipid binding pockets, including phosphatidic acid binding proteins (( Fig. 6D) (42,43)). When mapped onto the structure of the tandem BRCT domains, these residues were solvent-accessible and, therefore, potentially able to participate in lipid binding (Fig. 6E).
To experimentally test this hypothesis, we purified recombinant GST-BRCT and performed a lipid strip overlay experiment. The GST-RING domain was used as a negative control.
GST-BRCT was capable of binding several lipids, including phosphatidic acid, phosphatidyl inositol-3,5-P 2 and phosphatidyl inositol-4,5-P 2, whereas GST-RING was unable to bind to any lipids (Fig. 6F). Therefore, the BRCT domain of BRCA1 has a previously uncharacterized lipid binding activity that may mediate constitutive localization at ER membranes. Constitutive BRCA1 association with the ER may allow for rapid recruitment to the IP 3 R upon apoptotic induction. Of note, clinically relevant mutations of residues within the BRCT domain alter the subcellular distribution of BRCA1, which may be related to the lipid binding activity of the BRCT domain (44). It is also  Cells were harvested and fractionated using differential centrifugation as in Fig. 1C. 20 g of each fraction was used for Western blotting with anti-BRCA1 antibody (top row). IP 3 R antibody was used to show the purity of the ER-enriched fraction (center row). LRP130, a mitochondrial marker, was used to show the absence of mitochondrial proteins in the microsomal ER-enriched fraction (bottom row). The loss of LRP130 from the mitochondrial fraction after paclitaxel treatment may be related to its putative role in Taxol-mediated cell death signaling (50,51). For simplicity, only the mitochondrial and ER fractions are shown. The amount of BRCA1 protein present in the ER (100,000 ϫ g) fraction was quantified and graphed from three separate experiments and is presented as the -fold increase over basal (no paclitaxel). p Ͻ 0.05. C, positional score of amino acid residues in BRCA1 that have homology to 1185 fatty acid binding protein-specific PSSM libraries (see "Experimental Procedures"). A high-scoring region is present in the first BRCT repeat encompassing amino acids 1664 -1696 (solid line). The relative positions of domains in BRCA1 are shown above the graph. D, amino acids encompassing the putative lipid binding motif. Basic residues and adjacent threonine residues found in some lipid binding pockets (42,43)  possible that the BRCA1 lipid binding domain may interact with other cellular membranes in addition to the ER. Although this may indeed be the case and may have functional relevance, with respect to this study we have shown that BRCA1 is recruited to the IP 3 R at ER membranes during apoptosis to mediate apoptotic calcium release (Fig. 5) and, further, that BRCA1 retention at the ER is reduced in IP 3 R null cells (Fig. 6,  A and B). Therefore, at least some of the proapoptotic activities of BRCA1 are mediated by ER localization and binding to IP 3 R channels.

DISCUSSION
In this study, we established for the first time a possible mechanism through which BRCA1 exerts its proapoptotic function. Our data suggests a physical and functional interaction between BRCA1 and IP 3 R-1 that increases IP 3 R activity. We showed that BRCA1 is recruited to the ER during apoptosis and that loss of IP 3 R expression abolishes this recruitment. Finally, we identified a novel lipid binding activity in BRCA1 with significant implications for BRCA1 function. Future work will investigate the lipid binding activity of BRCA1 in more detail.
Although BRCA1 mutations and loss of expression are considered contributing causes of hereditary breast and ovarian cancer, studies have suggested that BRCA1 down-regulation may be present in a large number of breast tumors without mutations in BRCA1 (45)(46)(47). Furthermore, it has been shown that cytosolic localization of BRCA1 can be correlated with a better disease prognosis (48). We suggest that the proapoptotic function of BRCA1 is to increase IP 3 R-mediated apoptotic calcium release. This has significant implications for both hereditary and non-hereditary breast and ovarian cancers because BRCA1 expression mediates cellular responses to chemotherapeutics (40). The addition of yet another tumor suppressor, BRCA1, as a regulator of IP 3 Rs adds even more importance to the need to understand the role of calcium homeostasis in tumor progression.